Molecular Probes proposes to develop improved fluorophores on which to base fluorescent enzyme substrates. The fluorophores will have higher sensitivity than those in current use and are designed to have lower interference from sample autofluorescence. Some of the fluorophores will have spectral properties designed to permit use of low cost long-wavelength excitation sources. New ways of quenching background fluorescence from the substrate will be investigated as will new methods for development and localization of fluorescence in enzymatic products. The intent is to first improve the spectral properties of the dyes used as a basis of substrates and to devise unique methods using fluorescent substrates then to apply these new fluorophores and methods to preparation of substrates for disease detection and for cellular studies. Collaborative research will be supported in several areas utilizing new substrates such as monitoring gene incorporation, enzyme development, cell viability and tracing, receptor mediated endocytic processes and improved materials and processes for flow cytometric enzymatic analysis.